Upon damage to a blood vessel, subendothelial structures are exposed that mediate platelet adhesion through interaction with von Willebrand factor (vWF). vWF forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib (gpIb), an interaction especially important under high shear conditions, leading to the formation of a haemostatic plug and thus preventing excessive bleeding (Bennett S, Thromb Haemost (2001) March; 85(3):395-400). During normal homeostasis, these processes lead to wound healing of the damaged blood vessel wall. In pathological conditions however, excessive platelet function may lead to thrombus formation. The vWF subunit is composed of several homologues domains each covering different functions. vWF interacts through its A3 domain with fibrillar collagen fibers and through its A1 domain with the platelet receptor gpIb. Under normal conditions platelets and vWF do not interact. However, when vWF is bound to collagen at high shear rate, it is believed to undergo a conformational change allowing its binding with the platelet receptor gpIb. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion through the collagen receptors on the platelets (gpIa/IIa, gpVI, gpIV, p65, TIIICBP) resulting in platelet activation. This leads to activation of the gpIIb/IIIa receptor, fibrinogen binding, and finally to platelet aggregation.
Platelet aggregation inhibitors have been isolated from blood sucking organisms such as leech. Saratin, derived from leech Hirudo medicinalis is described in WO 02/15919 A2 and in Cruz C P et al ref. Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model. Journal of Vascular Surgery, 2001, 34: 724-729 and in Smith T P et al, Saratin, an inhibitor of collagen-platelet interaction, decreases venous anastomotic intimal hyperplasia in a canine dialysis access model, Vasc Endovascular Surg. 2003 July-August; 37(4):259-69.
Antibody-based therapeutics have been developed, some of which are currently used in therapy.
Abciximab (Chimeric 7E3 Fab; ReoPro; U.S. Pat. No. 6,071,514, EP 0 882 453), the Fab fragment of the mouse human chimeric antibody 7E3 which inhibits ligand binding to the platelet gpIIb/IIIa receptor, was approved for human use as adjunctive therapy to prevent ischemic complications of percutaneous coronary interventions in December 1994. The principle safety issue with gp IIb/IIIa inhibitors is the risk of bleeding, as the potent anti-platelet effect of these drugs may adversely affect homeostasis.
A murine monoclonal antibody was developed against vWF A1 domain (US 2002/0028204 A1; U.S. Pat. No. 6,280,731 and in WO 00/10601) and against its active conformation (U.S. Pat. No. 6,251,393). The in vivo efficacy is described in Kageyama S, et al: “Effect of a humanized monoclonal antibody to von Willebrand factor in a canine model of coronary arterial thrombosis”, Eur J Pharmacol. 2002 May 17; 443(1-3):143-9, and in “Anti-human vWF monoclonal antibody, AJvW-2 Fab, inhibits repetitive coronary artery thrombosis without bleeding time prolongation in dogs”. Thromb Res., 2001 Mar. 1; 101(5):395-404. and in “Anti-human von willebrand factor monoclonal antibody AJvW-2 prevents thrombus deposition and neointima formation after balloon injury in guinea pigs”. Arterioscler Thromb Vasc Biol. 2000 October; 20(10):2303-8). AJvW-2 inhibited high shear stress induced aggregation of human platelets and had no effect on low shear stress induced platelet aggregation.
The effects in baboons of a murine antibody 82D6A3 raised against the A3 domain of human vWF, are disclosed in WO 02/051351, and Dongmei Wu et al, “Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons”. Hemostasis, thrombosis and vascular biology, 2002, 99: 3623-3628.
Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human gpIb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. When injected into baboons, intact IgG and its F(ab′)(2) fragments caused almost immediate thrombocytopenia, due to the bivalency of F(ab′)(2) which mediates platelet crosslinking, or Fc:Fc receptor interactions which mediate activation of platelet aggregation (WO 0110911; Cauwenberghs N. et al, Arteriosclerosis, Thrombosis and Vascular biology, 2000, 20: 1347 and see, for example, Cadroy Y et al, Blood, 1994, 83: 3218-3224, Becker B H et al, Blood, 1989, 74: 690-694, Ravanat C. et al, Thromb. Haemost. 1999, 82: 528a abstract). Platelet deposition onto collagen-rich bovine pericardium was inhibited when Fab fragments were injected into the baboons before a thrombus was generated. However, when the Fab fragments were injected after a thrombus was allowed to form, no inhibition of further thrombosis was observed. The yields of expression of said Fab molecules are very low and the method of production is very labour intensive.